Tuberculosis of the eye in Italy: a forgotten extrapulmonary localization.

PURPOSE: Tuberculosis (TB) of the eye is a well-known extrapulmonary localization in high-incidence countries. Data on its relevance in developed countries are scanty. We aim to study the epidemiological and clinical pattern of ocular TB in a tertiary care institution of a western country. METHODS: From 2007 to 2010, consecutive patients with a diagnosis of isolated ocular TB or associated to extraocular TB were recruited. Patients with ophthalmological and clinical features of TB were treated with standard antitubercular therapy (ATT) and steroids in case of concomitant severe ocular inflammation. RESULTS: Seventeen cases of ocular and extraocular TB and 45 cases of isolated ocular TB were identified. The proportion of patients with ocular and extraocular TB in our local district was 8.1 %, with a proportion of 10.6 % for the isolated cases. In Cohort 1, only one patient was symptomatic for ocular impairment, and uveitis without inflammation was the most common presentation. On the contrary, in Cohort 2, all patients had visual impairment, mainly with bilateral involvement. 77.8 % of the patients showed an inflammatory pattern. ATT was administered for at least 9 months, in four cases with a short course of systemic corticosteroids. Eight cases in Cohort 2 showed recurrence after 1 year from diagnosis. CONCLUSIONS: TB of the eye should not be forgotten, even in geographical areas not considered among endemic countries. Ocular evaluation is advisable in patients with pulmonary and extrapulmonary TB, as early detection may allow ATT to preserve visual acuity.

PARP1 allows proper telomere replication through TRF1 poly (ADP-ribosyl)ation and helicase recruitment.

Telomeres are nucleoprotein structures at eukaryotic chromosome termini. Their stability is preserved by a six-protein complex named shelterin. Among these, TRF1 binds telomere duplex and assists DNA replication with mechanisms only partly clarified. Here we found that poly (ADP-ribose) polymerase 1 (PARP1) interacts and covalently PARylates TRF1 in S-phase modifying its DNA affinity. Therefore, genetic and pharmacological inhibition of PARP1 impairs the dynamic association of TRF1 and the bromodeoxyuridine incorporation at replicating telomeres. Inhibition of PARP1 also affects the recruitment of WRN and BLM helicases in TRF1 containing complexes during S-phase, triggering replication-dependent DNA-damage and telomere fragility. This work unveils an unprecedented role for PARP1 as a "surveillant" of telomere replication, which orchestrates protein dynamics at proceeding replication fork.

Locomotion performance for oscillatory swimming in free mode.

Oscillatory swimming of a fishlike body, whose motion is essentially promoted by the flapping tail, has been studied almost exclusively in axial mode under an incoming uniform stream or, more recently, self-propelled under a virtual body resistance. Obviously, both approaches do not consider the unavoidable recoil motions of the real body which have to be necessarily accounted for in a design procedure for technological means. Actually, once combined with the prescribed kinematics of the tail, the recoil motions lead to a remarkable improvement on the resulting swimming performance. An inviscid impulse model, linear in both potential and vortical contributions, is a proper tool to obtain a deeper comprehension of the physical events with respect to more elaborated flow interaction models. In fact, at a first look, the numerical results seem to be quite entangled, since their trends in terms of the main flapping parameters are not easy to be identified and a fair interpretation is obtained by means of the model capability to separate the effects of added mass and vortex shedding. Specifically, a prevailing dependence of the potential contribution on the heave amplitude and of the vortical contribution on the pitch amplitude is instrumental to unravel their combined action. A further aid for a proper interpretation of the data is provided by accounting separately for a geometrical component of the recoil which is expected to follow from the annihilation of any spurious rigid motion in case no fluid interactions occur. The above detailed decomposition of the recoil motions shows, through the numerical results, how the single components are going to influence the main flapping parameters and the locomotion performance as a guide for the design of biomimetic swimmers.

Expression of the soluble vascular endothelial growth factor receptor-1 in cutaneous melanoma: role in tumour progression.

BACKGROUND: Vascular endothelial growth factor (VEGF)-A, placenta growth factor (PlGF) and their corresponding membrane receptors are involved in autocrine and paracrine regulation of melanoma growth and metastasis. Besides the membrane receptors, a soluble form of the VEGF receptor (VEGFR)-1 (sVEGFR-1) has been identified, that behaves both as a decoy receptor, sequestering VEGF-A and PlGF, and as an extracellular matrix (ECM) molecule, promoting endothelial cell adhesion and migration through the interaction with α5ß1 integrin. OBJECTIVES: To analyse whether sVEGFR-1 plays a role during melanoma progression. METHODS: sVEGFR-1 expression was evaluated in a panel of 36 melanoma cell lines and 11 primary human melanocyte cultures by quantitative real-time polymerase chain reaction analysis and in specimens of primary or metastatic melanoma lesions from 23 patients by immunohistochemical analysis. RESULTS: sVEGFR-1 expression was highly upregulated in melanoma cell lines with respect to human melanocytes. Interestingly, cell lines obtained from cutaneous metastases showed a significant reduction of sVEGFR-1 expression, as compared with cell lines derived from primary tumours. These results were confirmed by immunohistochemical analysis of sections from primary skin melanomas and the corresponding cutaneous metastases, suggesting that modulation of sVEGFR-1 expression influences ECM invasion by melanoma cells and metastasis localization. Moreover, we provide evidence that adhesion of melanoma cells to sVEGFR-1 is favoured by the activation of a VEGF-A/VEGFR-2 autocrine loop. CONCLUSIONS: Our data strongly suggest that sVEGFR-1 plays a role in melanoma progression and that low sVEGFR-1/VEGF-A and sVEGFR-1/transmembrane VEGFR-1 ratios might predict a poor outcome in patients with melanoma.

Chagas disease in Italy: breaking an epidemiological silence.

Chagas disease, a neglected tropical disease that due to population movements is no longer limited to Latin America, threatens a wide spectrum of people(travellers, migrants, blood or organ recipients,newborns, adoptees) also in non-endemic countries where it is generally underdiagnosed. In Italy, the available epidemiological data about Chagas disease have been very limited up to now, although the country is second in Europe only to Spain in the number of residents from Latin American. Among 867 at-risk subjectsscreened between 1998 and 2010, the Centre for Tropical Diseases in Negrar (Verona) and the Infectious and Tropical Diseases Unit, University of Florence found 4.2% patients with positive serology for Chagas disease (83.4% of them migrants, 13.8% adoptees).No cases of Chagas disease were identified in blood donors or HIV-positive patients of Latin American origin. Among 214 Latin American pregnant women,three were infected (resulting in abortion in one case).In 2005 a case of acute Chagas disease was recorded in an Italian traveller. Based on our observations, we believe that a wider assessment of the epidemiological situation is urgently required in our country and public health measures preventing transmission and improving access to diagnosis and treatment should be implemented.

Impact of clinical data on chest radiography sensitivity in detecting pulmonary abnormalities in immunocompromised patients with suspected pneumonia.

PURPOSE: Chest radiography (CXR) of immunocompromised patients has low sensitivity in the early evaluation of pulmonary abnormalities suspected to be infectious. The purpose of the study was to evaluate whether the knowledge of clinical data improves the diagnostic sensitivity of CXR in the particular setting of immunocompromised patients after hematopoietic stem cell transplantation (HSCT). MATERIALS AND METHODS: Sixty-four CXRs of immunocompromised patients with clinically suspected pneumonia were retrospectively and independently evaluated by two radiologists to assess the presence of radiological signs of pneumonia, before (first reading) and after (second reading) the knowledge of clinical data. A chest computed tomography (CT) performed within 3 days was assumed as the standard of reference. For each reading, sensitivity of both radiologists was calculated. RESULTS: Readers showed a sensitivity of 39% and 58.5% for the first reading, and 43.9% and 41.5% for the second reading, respectively. For both readers, these values were not significantly different from those obtained at first reading (McNemar's test, p>0.05). Interobserver agreement at second reading was fair (Cohen test, k=0.33). CONCLUSIONS: The sensitivity of CXR is too low to consider it a stand-alone technique for the evaluation of immunocompromised patients after HSCT with suspected pneumonia, even if the radiologist knows detailed clinical data. For these patients, an early chest CT evaluation is therefore recommended.

The proteasome as a druggable target with multiple therapeutic potentialities: Cutting and non-cutting edges.

Ubiquitin Proteasome System (UPS) is an adaptable and finely tuned system that sustains proteostasis network under a large variety of physiopathological conditions. Its dysregulation is often associated with the onset and progression of human diseases; hence, UPS modulation has emerged as a promising new avenue for the development of treatments of several relevant pathologies, such as cancer and neurodegeneration. The clinical interest in proteasome inhibition has considerably increased after the FDA approval in 2003 of bortezomib for relapsed/refractory multiple myeloma, which is now used in the front-line setting. Thereafter, two other proteasome inhibitors (carfilzomib and ixazomib), designed to overcome resistance to bortezomib, have been approved for treatment-experienced patients, and a variety of novel inhibitors are currently under preclinical and clinical investigation not only for haematological malignancies but also for solid tumours. However, since UPS collapse leads to toxic misfolded proteins accumulation, proteasome is attracting even more interest as a target for the care of neurodegenerative diseases, which are sustained by UPS impairment. Thus, conceptually, proteasome activation represents an innovative and largely unexplored target for drug development. According to a multidisciplinary approach, spanning from chemistry, biochemistry, molecular biology to pharmacology, this review will summarize the most recent available literature regarding different aspects of proteasome biology, focusing on structure, function and regulation of proteasome in physiological and pathological processes, mostly cancer and neurodegenerative diseases, connecting biochemical features and clinical studies of proteasome targeting drugs.

Evaluation of biological and antimicrobial properties of freeze-dried whey fermented by different strains of Lactobacillus plantarum.

The aim of this study was to evaluate the biological and antimicrobial activities of commercial freeze-dried whey fermented by lactic acid bacteria in order to valorize this high polluting liquid waste of the dairy industry. Freeze-dried whey was fermented by different strains of Lactobacillus plantarum (CECT 220, 221, 748) at three different times of fermentation (24, 48, 72 h). Afterwards, the extract was purified on centricon amicon with a cut-off of 3 kDa to obtain a permeate consisting of small bioactive compounds reported in the literature to show greater bioactivity. The purified and diluted samples were subjected to the biological and antimicrobial tests for the evaluation of antioxidant, antihypertensive, iron binding, and antifungal activities and identification of phenolic compounds. The results highlighted a radical cation scavenging activity ranging from 1.415 to 2.083 mmol trolox equivalents TE per kg of dry weight, a percentage of iron binding capacity ranging between 23-55% and a percentage of ACE inhibitory activity ranging between 67-85%. The optimal biological activity was obtained from whey fermented by L. plantarum 220 for all the assays performed, except for the iron chelating activity. Furthermore, the antifungal analysis showed a good activity against the mycotoxigenic fungi belonging to Fusarium generum (F. moniliformis, F. graminearum and F. verticillioides), while a slight activity was obtained for Aspergillus and Penicillium generum. This antifungal activity could be correlated to the production of phenolic compounds during fermentation. The obtained results support the hypothesis of using whey as a functional ingredient to improve food preservation.

Anti-TNF-alpha therapy in seven patients with Behcet's uveitis: advantages and controversial aspects.

Behcet's disease (BD) is a chronic, relapsing, multisystem disease. In some patients, ocular involvement can lead to severe vision impairment despite immunosuppressive therapy. Since high levels of circulating TNF-alpha have been found both in peripheral blood and aqueous humor of patients with active BD, we evaluated the efficacy of anti-TNF-alpha therapy in seven patients with severe ocular involvement resistant to previous treatment. Seven patients with sight-threatening relapsing uveitis refractory to immunosuppressive regimens received intravenously infliximab, at a dose of 3-5 mg/kg, on week 0-2-4 and then every 6-8 weeks, in combination with low-dose prednisone and methotrexate or azathioprine. Efficacy was assessed in terms of number and severity of relapses of posterior uveitis, visual acuity, and reduction of corticosteroids and immunosuppressive drugs. After a mean follow-up period of 23 months, the total number of relapses dropped to 6, compared to the 21 observed in an equivalent period of time before treatment. The visual acuity improved in 4 eyes, while it remained stable in 9. Therapy with infliximab considerably reduced the required daily dose of both corticosteroids and immunosuppressive drugs. In our experience infliximab proved to be safe and effective in controlling both the number and intensity of cases of posterior uveitis and the extraocular manifestations of BD. It also allowed a reduction of corticosteroids and immunosuppressive drugs required to control the disease. However, ocular and systemic manifestations tended to recur after drug withdrawal or when the interval between infliximab courses was longer than 8 weeks. Moreover, infliximab administration is costly and requires hospital admission.

[Pathophysiology and prevention of contrast-induced acute renal failure]. / Fisiopatologia e prevenzione della insufficienza renale acuta da mezzo di contrasto.

Although the infusion of iodinated contrast media in diagnostic and interventional procedures may cause acute renal failure (ARF) especially in older or diabetic patients with preexisting nephropathy, these procedures are often unavoidable. Contrast medium-induced ARF is defined as an increase in serum creatinine of 0.5 mg/dL or a 25% or greater relative increase from baseline within 72 hours of iodinated contrast medium infusion. Because it is often very difficult to employ alternative diagnostic procedures, it is mandatory to adopt prophylactic protocols to prevent radiocontrast nephropathy. Renal hemodynamic lesions leading to medullary hypoxia, oxygen free radicals inducing tubular cell alterations, and parenchymal vasoconstriction are the main factors in the pathogenesis of contrast-induced ARF. Among the many proposed protocols to prevent contrast-induced renal toxicity, the most effective procedure is hydration with 1 mL/kg/h of isotonic saline solution in the 12 hours before and after contrast medium infusion. Promising results in terms of cardiac and renal protection have been reported in a recent trial with the use of high-dose N-acetylcysteine acting as an oxygen free radical scavenger: an intravenous bolus of 1200 mg N-acetylcysteine was given before coronary angiography followed by 1200 mg orally twice a day for 48 hours after the procedure. The protective effect seemed to involve not only the kidney: the drug was found to induce a significant reduction of the necrotic area in myocardial infarction.

[Sepsis, acute renal failure and multiple organ dysfunction syndrome]. / Sepsi, insufficienza renale acuta e "multiple organ dysfunction syndrome".

The so-called systemic inflammatory response syndrome (SIRS ) represents the cellular inflammatory and neuroendocrine systemic reaction in response to many adverse events. epsis is defined as IR induced by bacterial, mycotic or viral toxins. The circulating toxins deriving from the bacterial wall can activate the septic cascade that induces many systemic reactions involving the activation of the cellular immunity, complement and coagulation system. The endothelial cell is the target of the systemic phlogistic reaction; its stimulation is followed by the production of many vasoactive paracrine and systemic agents. In this context, local and systemic cytokine production plays a major role in inducing the septic cascade, which although meant to be a phlogistic defense reaction, can often become an uncontrolled and dangerous inflammatory reaction. The sepsis-derived lesions can involve many organs and apparatus leading to the picture of sepsis syndrome. Sepsis syndrome often induces severe pulmonary lesions with a picture of acute respiratory distress syndrome (ARDS ). The combination of acute renal failure and sepsis is associated with a high mortality rate, namely in patients with a nitric oxide-induced systemic reduction in peripheral vascular resistances and septic shock. The toxinemia can also induce myocardial damage with a reduction in cardiac performance. Therefore, septic patients who have a combination of pulmonary, cardio-vascular, renal and cerebral lesions present with the picture of multiple organ dysfunction syndrome, that can increase mor-tality to > 0%.

[Polymyxin-B direct hemoperfusion (PMX-DHP) in gram negative sepsis]. / L'moperfusione diretta con Polimixina-B (PMX-DHP) nel trattamento della sepsi da gram negativi.

UNLABELLED: Severe sepsis and septic shock have a mortality rate that may range between 28 and 50%. It is estimated that approximately 200,000 patients die per annum in the USA as a consequence of sepsis. The reduction of plasma endotoxin levels to achieve a favourable outcome for septic patients has been previously demonstrated but the effectiveness of treatments targeting single inflammatory mediators during established sepsis has been disappointing. Furthermore,some clinical study clinically showed valuable reduction in cytokine levels by hemofiltration alone. The prompt removal of endotoxins could be an effective way to reduce the immunological activation and the amount of NO produced by endotoxin-activated inducible NO-synthase in many tissues and cells. The polymyxin B cartridge is an extracorporeal hemoperfusion device (PMX-DHP) known to remove circulating endotoxins. Open-label clinical trials testing PMX-DHP have demonstrated its safety in the septic shock treatment while the overall survival rate significantly improved in comparison with the control groups. The purpose of this study was to investigate the effects of PMX-DHP on redox status, inflammatory cytokine profile, monocytes and PMN leukocyte activation in Gram-negative sepsis. Prospective study: six patients, 2 males and 4 females 60.5+/-24.5 years old, in ICU for severe Gram-negative sepsis (emergency surgery for intra abdominal infection). Two PMX-DHP runs, at T0 and T1; 2 hours each; the first within 24 hours from sepsis diagnosis or 12 hours after emergency surgery, the first PMX-DHP at T0, the second after 24 hours.; APACHE II score at T0: 20.1+/-3.7; SOFA score 14.2+/-2.5; organ failure: 3+/-1.5; norepinephrine(Ne) in 1 patient; Ne + dopamine (DA) in 4 patients; DA in 1 patient only. Mean dosage: Ne 0.24 mcg/kg/min; DA 8.9 mcg/kg/min. Four patients in CRRT (continuous veno-venous hemofiltration, AN69 hemofilter) for the entire length of the study. QB 100+/-10 ml/min. Pre and post PMX-DHP, plasma endotoxins as well as anti-IL 1-beta, IL2, IL4, IL5, IL6, IL8, IL10, TNF-alpha, GM-CSF, IFN-gamma levels were measured. Expression of CD64 on monocytes and PMN leukocytes and I -2r CD25 on CD4+ T cells by flow cytometry. Total and reduced plasma cysteine, homocysteine, glutathione (GSH); plasma glutathione peroxidase (GSH-Px) and reductase (GSH-Rx); erythrocyte GSH (eGSH), eGSH-Px and eGSH-Rx; NADP and NADPH and their ratio assessed pre and post PMX-DHP, all compared with 15 age and gender-matched healthy subjects for complete REDOX characterization. RESULTS: We observed a significant reduction of endotoxin levels post PMX-DHP; CD64 monocytes and PMN leukocytes overexpression returned to normal; pro-inflammatory cytokines Il6, Il 10 and TNF-alpha were significantly reduced. We detected no differences in plasma levels of anti-IL 1-beta, IL2, IL4, IL5, IL8, GM-CSF, IFN-gamma pre versus post PMX-DHP. SOFA score from 14.2+/-2.5 to 8.9+/-2.1 post PMX-DHP runs. Four out of six patients survived and were discharged; mortality was 33% versus the anticipated 51%. CONCLUSION: PMX-DHP reduces circulating endotoxins, down-activates monocytes and PMN leukocytes, reduces pro-Inflammatory cytokines and corrects the redox environment imbalance preventing oxidative damage to endothelial cells and the metabolic and functional microvascular derangements that usually lead to multi-organ failure and septic shock.

O6-alkylguanine-DNA alkyltransferase attenuates triazene-induced cytotoxicity and tumor cell immunogenicity in murine L1210 leukemia.

Methylating and chloroethylating triazene compounds (TZCs) are effective antitumor agents in murine leukemias and can induce the appearance of novel antigens in leukemic cells (chemical xenogenization). Recently, it has been shown that TZCs might have a role in the treatment of patients affected by acute myelogenous leukemias that express low levels of the DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase (OGAT). In this report, we have evaluated the role of this DNA repair enzyme in the leukemic cell response to the xenogenizing and cytotoxic properties of TZCs. OGAT-deficient murine leukemic L1210 cells were transfected with a recombinant ecotropic retrovirus containing the coding region for the human OGAT protein. Selected clones expressed the human OGAT transcript and had greatly increased OGAT activity. Compared to OGAT-deficient cells, OGAT-expressing cells were considerably more resistant to the xenogenizing properties of 1-(p-chlorophenyl)-3,3- dimethyl-triazene, measured in terms of leukemia graft rejection, and were less susceptible to the cytotoxic activity of the TZCs 8-carbamoyl-3-methyl-imidazo [5,1-d]-1,2,3,5-tetrazin-4(3H)-one and 8-carbamoyl-3-(2-chloroethyl)imidazo [5,1-d]-1,2,3,5-tetrazin-4(3H)-one. These data suggest that methylation of the O6 position of guanine is involved in the appearance of increased tumor immunogenicity after exposure to methylating TZC and that OGAT is able, at least in part, to counteract the cytotoxic effects of methylating and chloroethylating agents.

Alternative splicing generates at least five different isoforms of the human basic-FGF receptor.

Fibroblast growth factors (FGFs) are polypeptide mitogens that induce the proliferation of a wide variety of cell types. Of the seven family members, the best characterized are basic and acidic FGF. In addition to their mitogenic effects, they participate in angiogenesis, differentiation and maintenance of survival of neurons, cell migration and embryonal development. Of all family members, keratinocyte growth factor (KGF) is unique in that it is a specific mitogen for epithelial cells and does not interact with the FGF receptor of fibroblasts. To study the interactions between KGF and its receptor, we isolated KGF and FGF receptors from keratinocytes and fibroblasts, respectively. In the course of this study, we isolated five different variants of the FGF receptor from human fibroblasts and showed that all were derived from a single genetic locus. Four of these variants encode transmembrane receptors and can be divided into two subgroups that differ from one another with respect to the number (two or three) of immunoglobulin (Ig)-like domains. Within each subgroup, one receptor differed from the other by the presence of a two-codon insertion. Thus, all the variations among the four isoforms are localized to their ligand binding domains. The fifth isoform encodes a molecule truncated just 3' to the first Ig-like domain and thus could be secreted from the cell. The transcripts encoding the long and short isoforms were found to be expressed in many cell types, but their relative levels of expression varied greatly depending on the cell type. These findings indicate that alternative splicing generates diverse FGF receptor isoforms in human cells.

The N-terminal region of proto-dbl down regulates its transforming activity.

The dbl proto-oncogene can transform NIH3T3 cells when overexpressed, but its transforming activity is about 50 to 70 fold lower than that of the dbl oncogene. The dbl oncogene encodes a protein of 478 amino-acids while proto-dbl encodes a protein of 925 amino-acids. The genesis of dbl involved the loss of the first 497 amino-acids of proto-dbl and the acquisition of a new N-terminus from another human locus. The last 428 amino-acids of proto-dbl and dbl product are identical with the exception of a single conservative amino-acid change. Any of these alterations could be responsible for the greater transforming activity of dbl. In order to define the role of these alterations more precisely, we constructed two deletion mutants, one derived from proto-dbl and the second from dbl in which only their last 428 amino-acids were retained. Under the control elements of the same promoter, the transforming activity of each of these mutants was similar to that of the dbl oncogene, i.e. 60-80 fold greater than that of proto-dbl. This finding suggests that the loss of the first 497 amino-acid of proto-dbl, rather than the acquisition of a new N-terminus, is crucial to the enhanced transforming activity of the dbl oncogene. Both mutant proteins were equally distributed between the membrane and cytosolic fractions, a pattern similar to that of their corresponding parental proteins. These results suggest that the subcellular distribution of proto-dbl is determined by its C-terminal 428 amino-acids. Unlike their parental proteins, neither mutant was phosphorylated, indicating that phosphorylation is not required for dbl transforming activity. In addition to the lack of phosphorylation, each mutant protein had a half-life of 5-6 h while the half-life of proto-dbl was about 1 h. Thus, our data suggest that the N-terminal half of proto-dbl can down regulate its transforming activity and that sequences within this region are responsible for rapid turnover of the protein.

The human dbl-proto-oncogene product is a cytoplasmic phosphoprotein which is associated with the cytoskeletal matrix.

The translational product of the dbl oncogene is a 66 kDa (p66) protein with no apparent sequence similarity to any of the known oncogene products, whereas the human dbl proto-oncogene encodes a translational product of 115 kDa (p115). We compared proto-dbl p115 and dbl p66 with respect to their subcellular localization, biogenesis and post-translational modifications. Like p66, p115 was found to be a cytoplasmic phosphoprotein present in both cytosol and crude membrane preparations. Membrane fractionation studies revealed that p115 as well as p66 were primarily associated with fractions enriched in plasma membranes, suggesting that this subcellular compartment is a likely site of action of dbl proteins. The membrane-associated forms of p115 and p66 were fairly resistant to solubilization by nonionic detergents, suggesting that dbl proteins associate with the cytoskeletal matrix. p115 was also found to be phosphorylated primarily on serine residues. However, p115 was phosphorylated to a lesser extent as compared to the phosphorylated form of p66. The half-life of proto-dbl p115 was significantly shorter (1 hour) than that of dbl p66 (5-6 h). The higher stability of p66 is likely due to the acquisition of unrelated human sequences and/or to the deletion of the N-terminal region of proto-dbl.

Induction of Xenopus oocyte meiotic maturation by the dbl oncogene product.

The dbl oncogene was originally identified by transfection of NIH3T3 cells with DNA from a human diffuse B-cell lymphoma. The dbl oncogene product is a cytoplasmic phosphoprotein distributed between the cytosolic and cytoskeletal matrix-associated membrane fractions. Nucleotide sequence analysis has indicated that the predicted dbl product is very hydrophilic with no detectable similarity to known oncogene products. We have more recently discovered that a region of dbl essential for its transforming activity shows significant sequence similarity to a yeast cell cycle gene, CDC24, which is involved in cell polarity and bud formation in the division cycle of Saccharomyces cerevisiae. This sequence similarity suggests a possible role for dbl in cell division. We report here that the dbl oncogene product is able to induce maturation of Xenopus oocytes. Germinal vesicle breakdown (GVBD) was observed when oocytes were microinjected with the soluble fraction of SF9 insect cells infected with a dbl recombinant baculovirus as well as with the in vitro-transcribed dbl mRNA. Moreover, extracts of oocytes microinjected with dbl mRNA showed activation of H1 histone kinase activity. These findings define a new biologic activity of the dbl product and provide the opportunity to analyse dbl interactions with other components of signaling pathways involved in oocyte maturation.

Combined effects of adenovirus-mediated wild-type p53 transduction, temozolomide and poly (ADP-ribose) polymerase inhibitor in mismatch repair deficient and non-proliferating tumor cells.

Lack of p53 or mismatch repair (MR) function and scarce cell proliferation are commonly associated with tumor cell resistance to antineoplastic agents. Recently, inhibition of poly(ADP-ribose) polymerase (PARP) has been considered as a tool to overcome resistance of MR-deficient tumors to methylating agents. In the present study we demonstrated that infection with p53 expressing adenovirus (Ad-p53), enhances chemosensitivity of MR-deficient tumor cell lines to the methylating agent temozolomide (TZM), either used as single agent or, more efficiently, when combined with PARP inhibitor. Moreover, the association of Ad-p53 with drug treatment induced a more pronounced growth inhibitory effect than that provoked by Ad-p53 infection only. Cells, growth arrested by p53 transduction, and then subsequently exposed to the drugs, were still highly susceptible to cytotoxicity induced by TZM and PARP inhibitor. The results suggested that this drug combination might be effective even in non-proliferating tumor cells. It is conceivable to envisage future possible strategies to enhance cytostatic or cytotoxic effects induced by Ad-p53, based on the use of TZM, alone or combined with PARP inhibitor for the therapy of resistant tumors.